Depletion of IgG-producing plasma cells in the colon by treatment of proteasome inhibitor ameliorates chronic DSS-induced colitis in mice

Depletion of IgG-producing plasma cells in the colon by treatment of proteasome inhibitor ameliorates chronic DSS-induced colitis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Depletion of IgG-producing plasma cells in the colon by treatment of proteasome inhibitor ameliorates chronic DSS-induced colitis in mice Shin Ebihara, Yukari Kimoto, Rumi Katsumoto, Noriko Konishi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7646295/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract Basal plasmacytosis is a histopathological hallmark of ulcerative colitis (UC). However, the precise roles of plasma cells in UC pathogenesis remains unknown. In this study, we investigated the effects of a proteasome inhibitor, which depletes plasma cells, on chronic colitis progression in mice to clarify their contribution to disease pathogenesis. Chronic colitis was induced in female C57BL/6 mice by three cycles of ad libitum dextran sulfate sodium (DSS) feeding followed by distilled water (DW), each cycle lasting seven days. The proteasome inhibitor bortezomib was administered intravenously twice weekly for three weeks after the second DSS/DW cycle. Elevated plasma IgG and perinuclear anti-neutrophil cytoplasmic antibody (pANCA) levels, as well as infiltration of IgG-producing plasma cells into the colon, were observed after the second DSS/DW cycle in chronic DSS-induced colitis model mice. Plasma cells in the colon exhibited an immature CD19⁺CD138⁺ phenotype. Bortezomib significantly ameliorated colitis and intestinal fibrosis by reducing plasma IgG and pANCA levels and the number of IgG-producing plasma cells in the colon. In conclusion, IgG-producing plasma cells were involved in colitis pathogenesis, and their depletion ameliorated colitis. Thus, IgG-producing plasma cells are causative factors and potential therapeutic targets for UC. Health sciences/Diseases Health sciences/Gastroenterology Biological sciences/Immunology IgG-producing plasma cells chronic DSS-induced colitis model proteasome inhibitor pANCA ulcerative colitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease, is a group of chronic relapsing gastrointestinal tract disorders pathologically characterized by intestinal inflammation and epithelial injury [ 1 , 2 ]. Basal plasmacytosis, defined as dense infiltration of plasma cells around the deep part of the lamina propria (LP) or at the base of colon crypts, is a histological feature of IBD, particularly UC [ 3 , 4 ]. High degree of basal plasmacytosis is associated with high clinical relapse rate [ 5 , 6 ], shorter clinical relapse time [ 7 ], and elevated fecal calprotectin levels (disease activity markers) in patients with UC [ 8 ]. For patients with UC, several antibodies are used as serological markers [ 9 ]. Specifically, perinuclear anti-neutrophil cytoplasmic antibody (pANCA), an autoantibody, is the best marker for UC in terms of sensitivity and specificity [ 9 ]. Recently, autoantigens, such as integrin αvβ6 [ 10 ] and endothelial protein C receptor [ 11 ], have also been identified in patients with UC. Serum levels of IgG [ 12 ] and pANCA [ 13 ] positively correlate with the Mayo Clinic score, suggesting that IgG-producing plasma cells are associated with UC pathogenesis. Plasma cells comprised one of the major cellular components in the colonic LP of patients with UC [14 − 16]. In a healthy state, most plasma cells are IgA-producing plasma cells; however, marked IgG-producing plasma cell infiltration is observed in the colon of patients with UC [ 15 , 17 ]. IgG-producing plasma cells infiltrate the inflamed mucosa and critically influence UC pathogenesis by exacerbating mucosal inflammation by activation of pathogenic intestinal macrophages via Fc gamma receptor (FcγR) signaling [ 15 ]. Despite these insights, the precise roles of IgG-producing plasma cells in UC remain unclear [ 15 , 16 ]. Chronic dextran sulfate sodium (DSS)-induced colitis model is established by administering three repeated cycles of ad libitum of DSS followed by distilled water (DW) every seven days [ 18 , 19 ]. The chronic DSS-induced colitis model exhibits more resemblance to chronic UC than the acute DSS-induced colitis model in terms of the association with both myeloid cells, such as macrophages and neutrophils, and lymphocytes, such as T and B cells [ 20 ]. Therefore, the chronic colitis model is often used to evaluate the therapeutic efficacy of anti-inflammatory drugs for UC, such as glucocorticoids (dexamethasone) [ 21 ], Janus kinase (JAK) inhibitors (tofacitinib) [ 22 ], and anti-tumor necrosis factor (TNF)-α antibodies [ 21 , 23 ]. Bortezomib is a proteasome inhibitor that enhances the accumulation of unfolded proteins with the induction of endoplasmic reticulum (ER) stress, leading to apoptotic cell death [ 24 ]. Plasma cells synthesizing high levels of immunoglobulins exhibit relatively high sensitivity to bortezomib [ 25 ], contributing to the clinical success of bortezomib for multiple myeloma treatment [ 26 ]. Bortezomib ameliorates lupus nephritis [ 27 ], ANCA-induced glomerulonephritis [ 28 ], and myasthenia gravis (MG) [ 29 ] in rodents by inhibiting the production of autoantibodies by plasma cell depletion. Additionally, bortezomib protects against autoimmune diseases, such as systemic lupus erythematosus [ 30 ], thrombotic thrombocytopenic purpura [ 31 ], MG [ 32 ], and neuromyelitis optica spectrum disorder [ 33 ], by reducing the plasma cell number and suppressing autoantibody production. In this study, we established a chronic colitis model by repeated DSS treatment of mice and investigated the roles of IgG-producing plasma cells in colitis pathogenesis. Additionally, to demonstrate plasma cells as causative cells for chronic colitis, we examined the effects of bortezomib, which depletes plasma cells, in a colitis model. Results pANCA and anti-flagellin IgG are produced during intestinal inflammation in chronic DSS-induced colitis model mice Chronic colitis was induced by administering three repeated cycles of DSS. Each cycle involved the administration of 1.5% DSS for seven days, followed by sterile DW ad libitum for seven days (Fig. 1 A). In the DSS-induced colitis model, mice treated with DSS started to exhibit clinical signs of colitis, including weight loss with loose stools, diarrhea, and hematochezia, after five days of DSS administration during the first DSS/DW cycle. Body weight further decreased when DW was reintroduced on day 8, with maximal weight loss observed on day 10 (Fig. 1 B). Colitis model mice started to regain weight at the end of the first DSS/DW cycle (day 15) until the initial body weight was reached at the end of the experiment. As shown in Fig. 1 C, after the first DSS/DW cycle, during the course of acute intestinal inflammation, total IgG levels in the plasma were lower than those in DW-treated mice, consistent with a previous report [ 34 ]. However, after the second DSS/DW cycle during the chronic inflammation phase, total IgG levels were higher than those in DW-treated mice (Fig. 1 C). Next, we examined whether IgG against flagellin, protein subunit of bacterial flagellum, or myeloperoxidase (MPO), an autoantigen, were present in the chronic DSS-induced colitis model mice during intestinal inflammation. As shown in Fig. 1 D, although the plasma flagellin-specific IgG antibody levels increased only after the second DSS/DW cycle (day 29), consistent with previous reports [ 34 , 35 ], these antibody levels did not increase during the chronic phase. In contrast, pANCA (anti-MPO IgG) levels consecutively increased after the second DSS/DW cycle until the experimental endpoint (day 50; Fig. 1 E). Plasma IgA levels did not increase at any time point, indicating that they did not affect colitis development (Fig. 1 F). Taken together, these results suggest that autoantibodies, such as pANCA, are involved in colitis pathogenesis. IgG-producing plasma cells infiltrate into colonic LP in chronic DSS-induced colitis model mice Flow cytometric analysis revealed significantly higher numbers of CD4 T cells (TCRβ + CD335 − CD4 + ), B cells (CD19 + B220 + ), macrophages (CD11b + F4/80 + ), inflammatory monocytes (CD11b + F4/80 − Ly6C + ), and neutrophils (CD11b + Ly6G + ) in the colonic LP of DSS-treated mice than in that of DW-treated mice at the experimental endpoint (Fig. 2 A and Supplementary Fig. 1). Furthermore, consistent with the increase in total plasma IgG and pANCA production (Fig. 1 C and E), IgG-expressing CD19 + cells were observed in the mucosa and submucosa of the colon of DSS-treated mice at the experimental endpoint (Fig. 2 B). We confirmed the infiltration of CD19 + cells into the colonic LP of the chronic DSS-induced colitis model using flow cytometry. CD19 + cells infiltrating the colon were B220 + CD19 + B and B220 − CD19 + CD138 + immature plasma cells (also called plasmablasts; Fig. 2 C). Low CD138 levels in immature plasma cells are possibly due to its instability, which affects its detection during collagenase digestion [ 36 ]. Here, immature plasma cells expressed IgA and IgG2 (Fig. 2 C). In contrast, B cells mildly expressed IgA, but not IgG (Fig. 2 C). In addition to the various inflammatory cells, the number of immature plasma cells also increased in the colon of the chronic DSS-induced colitis model (Fig. 2 D). As shown in Fig. 2 E and F, both IgA- and IgG-producing immature plasma cells were more abundant in DSS-treated mice than in the DW-treated mice. Overall, plasma cells infiltrating the colon in the chronic DSS-induced colitis model were IgG2-producing and exhibited an immature phenotype. Effects of bortezomib on the inflammatory responses in chronic DSS-induced colitis model mice To investigate the role of colon-infiltrating IgG-producing immature plasma cells in the chronic colitis progression, immature plasma cells were depleted by systemic administration of bortezomib. For intervention studies, bortezomib was administered twice weekly to the mice at the end of the second DSS/DW cycle (Fig. 3 A). To explore the inhibitory effects of bortezomib, total IgG and pANCA levels in plasma, which were upregulated during colitis progression, were examined. Bortezomib significantly decreased the total IgG (Fig. 3 B) and pANCA (Fig. 3 C) levels at the experimental endpoint comparable to those at the start of treatment (pre-treatment). As shown in Fig. 3 D, the numbers of CD4 T cells, B cells, plasma cells, macrophages, inflammatory monocytes, and neutrophils increased at the start of bortezomib treatment. Moreover, the numbers of macrophages and neutrophils decreased, but those of other cells, excluding plasma cells, remained unchanged at the end of treatment in the vehicle group. Bortezomib significantly decreased the numbers of B and plasma cells, but not CD4 T cells, macrophages, inflammatory monocytes, and neutrophils, in the colonic LP (Fig. 3 D). As shown in Fig. 3 E and F, the numbers of IgG2- and IgA-producing immature plasma cells increased during colitis progression. Notably, bortezomib reduced the number of IgG2-producing immature plasma cells more than that of B or total plasma cells, but not IgA-producing immature plasma cells, in the colonic LP (Fig. 3 E and F). Moreover, IgG was deposited in the mucosa and submucosa of the colon in the chronic DSS-induced colitis model at the experimental endpoint, similar to that observed in patients with UC (Fig. 3 G) [ 37 ]. Bortezomib significantly decreased the colonic deposition of IgG (Fig. 3 G and H). Amelioration of colitis and intestinal fibrosis by bortezomib in chronic DSS-induced colitis model mice To investigate the effects of bortezomib on colitis pathogenesis and intestinal fibrosis, we performed histopathological evaluation of colitis (H&E staining) [ 38 ] and quantification of collagen deposition in the mucosa and submucosa of the colon (Masson’s trichrome staining) [ 39 , 40 ]. At the start of treatment with bortezomib (pre-treatment), the mice already exhibited colitis (Fig. 4 A and B) and mild intestinal fibrosis (Fig. 4 C and D). Bortezomib ameliorated colitis (Fig. 4 A and B) and inhibited intestinal fibrosis progression (Fig. 4 C and D). As shown in Supplementary Fig. 2A–C, bortezomib significantly reduced the colon weight-to-length ratio and colonoscopic disease severity, including mucosal thickening, vasculature, and granularity, at the experimental endpoint. As shown in Fig. 4 E, alpha-smooth muscle actin (αSMA) was localized not only in the typical tissue layers, such as muscolaris mucosa and muscolaris propria, but also in the mucosa of the chronic DSS-induced colitis model, similar to that observed in patients with UC [ 41 ]. Bortezomib decreased the αSMA levels in the colon at the experimental endpoint (Fig. 4 E). Along with the cell count results shown in Fig. 3 D–F, these findings suggest that bortezomib ameliorates colitis and the intestinal fibrosis progression by selectively depleting the IgG-producing plasma cells, without significantly affecting the other immune cells, including innate immune and CD4 T cells. Although the production of inflammatory cytokines, such as TNF-α, interleukin (IL)-6, and IL-1β, and nuclear factor (NF)-κB activation increased with the development of chronic colitis, bortezomib had no effects on these processes (Supplementary Fig. 3). Furthermore, anti-inflammatory agents targeting the innate immune or T cells, such as glucocorticoids (prednisolone), JAK inhibitors (tofacitinib), and anti-TNF-α antibodies, did not inhibit the development of colitis in the chronic DSS-induced colitis model (Supplementary Fig. 4). Therefore, IgG-producing immature plasma cells play crucial roles in the development and progression of colitis in the chronic DSS-induced colitis model. In summary, bortezomib significantly ameliorated chronic colitis by reducing the number of colonic IgG-producing immature plasma cells in the colon of the chronic DSS-induced colitis model. Discussion In a double-blind, randomized, placebo-controlled trial (phase II trial), anti-CD20 antibody rituximab did not induce remission in moderately active UC [ 42 ]. Rituximab depleted B cells, but not plasmablasts/plasma cells, in the colonic mucosa of patients with UC in a clinical trial, considering that plasmablasts/plasma cells remaining at the site of inflammation continuously supported the ongoing inflammatory process of UC [ 43 ]. In this study, we found that the chronic DSS-induced colitis model exhibits resemblance to the chronic nature of human UC in terms of histopathological features, including IgG-producing plasma cell infiltration into the colon. Interestingly, flow cytometric analysis showed that IgG-producing plasma cells infiltrating the colonic LP of chronic DSS-induced colitis model were immature plasma cells (also called plasmablasts), previously reported in patients with UC [ 15 , 44 , 45 ]. Antimicrobial antibodies are produced in the early stages, whereas autoantibodies are produced in the later stages of colitis. Therefore, colonic bacteria possibly contain proteins cross-reactive to the autoantigen epitope, resulting in a destructive inflammatory response triggered by the influx of luminal bacterial products into the gut wall via epithelial damage directed toward a self-antigen, leading to the progression from acute to chronic colitis in a colitis model, similar to that observed in patients with UC [ 46 , 47 ]. This study showed that bortezomib, a plasma cell-targeted proteasome inhibitor, significantly improved the colonoscopic and histopathological manifestations in the chronic DSS-induced colitis model. In addition to inducing ER stress, bortezomib inhibits NF-κB activation [ 48 ]. Bortezomib also inhibits the production of inflammatory cytokine, such as IL-6, TNF-α, and IL-1β, by suppressing NF-κB activation, thereby ameliorating acute DSS-inducing colitis [ 49 ]. However, bortezomib inhibited colitis progression without suppressing inflammatory cytokine production and NF-κB activation in the chronic-phase DSS-induced colitis model in this study. Bortezomib reduced not only plasma IgG levels but also number of IgG-producing immature plasma cells and IgG deposition in the colon of chronic DSS-induced colitis model mice. Therefore, bortezomib ameliorated chronic colitis by reducing the number of IgG-producing immature plasma cells in the colon. Interestingly, bortezomib did not decrease the number of IgA-producing immature plasma cells in the colon, suggesting differential activation of the ER stress-induced unfolded protein response between IgA- and IgG-producing immature plasma cells. As IgA production at mucosal sites is necessary to maintain a non-inflammatory state and prevent inflammatory responses [ 50 ], targeting IgG-producing immature plasma cells rather than IgA-producing immature plasma cells is important to prevent colitis. UC is associated with progressive fibrosis of the mucosa and submucosa linked to the severity and chronicity of inflammation [ 51 , 52 ], suggesting the importance of deep remission, including histological remission, as a therapeutic target for UC [ 53 ]. Intestinal fibrosis was also observed in our chronic DSS-induced colitis model, consistent with previous reports [ 39 , 40 ], was suppressed by bortezomib. Bortezomib significantly reduces lung fibrosis in bleomycin-treated mice by depleting the plasma cells, indicating the causal role of plasma cells in lung fibrosis development [ 54 ]. Thus, these findings suggest plasma cells as therapeutic targets for fibrosis, including lung and intestinal fibrosis. In contrast to previous reports [21 − 23], our chronic DSS-induced colitis model was resistant to treatment with glucocorticoids (prednisolone), JAK inhibitors (tofacitinib), and anti-TNF-α antibodies, possibly due to the variability in the microbiome among different facilities [ 55 ]. Therefore, our chronic DSS-induced colitis model was possibly a severe UC murine model that was not responsive to major therapeutic agents, as described above. Serum pANCA levels are associated with the lack of response to anti-TNF-α therapy in patients with UC [ 56 , 57 ]. Moreover, number of infiltrating plasma cells in the colon is higher in patients with UC non-responsive to anti-TNF agents [ 58 ] and glucocorticoids (prednisolone) [ 59 ] than in the responders. Therefore, plasma cell depletion may serve as an effective therapeutic approach for drug-resistant UC characterized by marked plasma cell infiltration into the colon. In conclusion, this study demonstrated the ameliorative effects of bortezomib, which depletes plasma cells, on chronic DSS-induced colitis. However, further translational research and clinical studies are necessary to assess the efficacy of bortezomib for UC treatment. Nevertheless, this study provides insights into the in vivo functions of IgG-producing immature plasma cells in the colonic LP of patients with UC. Our findings suggest that IgG-producing immature plasma cell depletion in the colon ameliorates chronic colitis, highlighting a novel therapeutic approach for human UC. Materials and method Mice Female C57BL/6J (B6J) mice (weighting 19 ± 2 g) were purchased from CLEA Japan (Tokyo, Japan) and maintained under specific pathogen-free conditions at a room temperature of 23 ± 3°C and air humidity of 55 ± 15% under a 12/12 h light/dark cycle. Chronic DSS-induced colitis model establishment and treatment Colitis was induced as previously described [ 18 , 19 ]. Briefly, mice were fed 1.5% DSS (36–50 kDa; MP Biomedicals, Solon, OH, USA) dissolved in sterile DW (Otsuka Pharmaceutical, Tokyo, Japan) ad libitum for seven days, followed by DW for seven days. This DSS/DW cycle was repeated twice, after which the mice were administered DW for seven days to establish a chronic DSS-induced colitis model. Sham group was treated with DW. Bortezomib was purchased from Santa Cruz (Dallas, TX, USA), dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) at a concentration of 10 mg/mL, and suspended in PBS. Female B6J mice were mixed and caged into groups of seven mice at ten days from the initial day based on their body weight (bw). Then, the mice were intravenously treated with the vehicle alone or bortezomib (750 µg/kg bw) twice weekly for three weeks from the end of the second DSS/DW cycle. In another experiment, after the second DSS/DW cycle, the mice were gavaged daily with another vehicle (0.5% methylcellulose (MC)), 3 mg/kg bw prednisolone or 100 mg/kg bw tofacitinib, or administrated intraperitoneally 1000 µg of anti-TNF-α antibody (clone TN3-19.12; Bio X cell, Lebanon, NH, USA) or Armenian hamster IgG (Bio X cell) twice a week. Colonoscopy and histology For colonoscopy, the mice were anesthetized with isoflurane (Viatris, Tokyo, Japan), and feces were removed by injecting saline through a flexible feeding tube (Fuchigami, Kyoto, Japan). A rigid telescope (diameter, 1.9 mm; Smith and Nephew, London, UK) was rectally inserted into the mice up to 3 cm using the Olympus CLH-250 Xenon Light Source (Olympus, Tokyo, Japan), as previously described [ 60 ]. During endoscope withdrawal, a video of the distal colon was recorded using the Olympus Video System OTV-SC2 (Olympus) and TEAC UR-4MD Medical Video Recorder (TEAC, Tokyo, Japan). Colonoscopic findings were scored as follows: Mucosal thickening (0 = transparent, 1 = moderate, 2 = marked, and 3 = non-transparent), vasculature (0 = normal, 1 = moderate, 2 = marked, and 3 = absent/bleeding), and granularity (0 = none, 1 = moderate, 2 = marked, and 3 = extreme) [ 60 ]. Colonoscopy findings were scored by a blinded reviewer. For microscopic examination, colons were removed within 24 h of endoscopy after euthanizing the mice via cervical dislocation under anesthesia with isoflurane (Viatris). Distal colons (3 cm) were longitudinally cut, fixed with 10% neutral-buffered formalin (FUJIFILM Wako Pure Chemical, Osaka, Japan), embedded in paraffin wax, and stained with hematoxylin and eosin (H&E). Tissue specimens for pathological evaluation were prepared by the Biopathology Institute (Oita, Japan). Distal colons were scored according to the histological criteria as follows: Inflammatory infiltration (0 = none, 1 = 50% transmural infiltration), epithelial hyperplasia (0 = none, 1 = 1.5-fold increase in epithelial length), goblet cell and crypt loss (0 = none, 1 = 50%), cryptitis (0 = none and 2 = presence), and crypt abscess (0 = none and 3 = presence) [ 38 ]. Histological scoring was supported by KAC (Kyoto, Japan). Measurement of plasma antibody levels Plasma samples were stored at − 80°C until use. Plasma IgG and IgA levels were determined using ELISA kits (Bethyl Laboratories, Montgomery, TX, USA), according to the manufacturer’s instructions. Levels of pANCA (anti-MPO IgG) and anti-flagellin IgG antibodies were detected by ELISA. Briefly, diluted plasma samples and standards (anti-MPO IgG (Abcam, Cambridge, UK) or anti-flagellin IgG (InvivoGen, San Diego, CA, USA)) were added to wells pre-coated with recombinant mouse MPO (R&D Systems, Minneapolis, MN, USA) or flagellin (FLA-ST; InvivoGen) and incubated at room temperature for 2 h. After washing with PBS/Tween 20, diluted horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and incubated for 2 h. After washing with PBS/Tween 20, TMB substrate was added to each well and incubated in the dark. Subsequently, the stop solution was added to each well, and absorbance was measured at 450 nm. Isolation of colonic LP cells LP cells were isolated from the colon, as previously described [ 60 ]. Briefly, distal colons were longitudinally cut, washed with cold Hank’s balanced salt solution containing 2% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 U/mL), and minced. The tissues were completely digested at 37°C for 40 min with gentle stirring in the RPMI-1640 medium containing 2% FBS, penicillin (100 U/mL), and streptomycin (100 U/mL) and supplemented with collagenase D (100 U/mL; Sigma-Aldrich, St Louis, MO, USA) and DNase (20 mg/mL; Roche Diagnostics, Basel, Switzerland). LP cells were purified on a 40/75% Percoll gradient via centrifugation at 600 × g for 20 min at 25°C. Flow cytometry Antibodies against CD45, TCRβ, CD11b, B220, CD19, CD138, and IgA were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against Ly6G, CD4, CD8b, CD11b, CD11c, F4/80, Ly6G, Ly6C, CD335 (NKp46), IgG1, IgG2a/b, and IgG3 were purchased from BioLegend (San Diego, CA, USA). Cell surface staining was performed according to standard techniques after treatment with the anti-CD16/32 antibody (BD Biosciences) to block FcγR binding. Dead cells were excluded using the Fixable Viability Dye eFluor 780 (FVD780; Thermo Fisher Scientific). For intracellular staining, the cells were stained with different cell surface antibodies, fixed, permeabilized, and intracellularly stained for IgA, IgG1, IgG2a/b, or IgG3. Gating strategies were set with reference to the isotype or fluorescence minus one control. Flow cytometry was performed using the BD LSRFortessa X-20 flow cytometer (BD Biosciences), and data were analyzed using the FlowJo version 10.8.0 software (BD Biosciences). Immunohistochemistry for IgG For CD19 and IgG staining, deparaffinized and rehydrated sections were treated with a protease solution (Nichirei Biosciences, Tokyo, Japan) for 15 min at 37°C and incubated with Blocking One (Nacalai Tesque) for 1 h at room temperature. Then, the sections were incubated with Alexa Fluor 488-conjugated anti-CD19 (BioLegend) and Alexa fluor 555-conjugated anti-IgG (BioLegend) at 4°C overnight. Nuclei were counterstanied using 4',6-diamidino-2-phenylindole (DAPI; SouthernBiotech, Birmingham, AL, USA). The sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the FV3000 confocal laser-scanning microscope (Olympus) for double-positive cells. For IgG deposits, the sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the BZ-X800 fluorescence microscope (Keyence, Osaka, Japan). Microscopic images were digitized, and IgG-positive areas were determined by red–green–blue (RGB) segmentation. Digital images were analyzed using inform version 3.1.2 software (PerkinElmer, Hopkinton, MA, USA). Results were calculated as the number of IgG-positive areas in the mucosa and submucosa in all fields of the distal colon. Colonic fibrosis analysis Masson’s trichrome staining was performed to measure the collagen deposition as fibrosis. The tissue specimens were prepared for pathological evaluation at the Biopathology Institute. Microscopic images were digitized, and collagen-positive areas were determined by RGB segmentation. Digital images were analyzed using the inform version 3.1.2 software (PerkinElmer). Results were calculated as the number of collagen-positive areas in the mucosa and submucosa in all fields of the distal colon. For αSMA staining, deparaffinized and rehydrated sections were treated with HISTOFINE (pH 9.0; Nichirei Biosciences) for 15 min at 95°C and incubated with Blocking One (Nacalai Tesque) for 30 min at room temperature. Then, αSMA staining was performed using rabbit anti-αSMA (Abcam) at 4°C overnight, followed by incubation with Alexa Fluor 647 anti-rabbit IgG (Abcam) at room temperature for 1 h. Nuclei were counterstained with DAPI (SouthernBiotech). The sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the BZ-X800 fluorescence microscope (Keyence). Measurement of colonic cytokine levels Distal colonic segments were rinsed with saline, blotted dry, and stored at − 80°C. The segments were homogenized using beads in DW containing the Sample Diluent Concentrate 1 (R&D Systems) and protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 12000 × g for 10 min at 4°C to remove the debris, protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA). Colonic cytokine levels were quantified using the Meso Scale Discovery electrochemiluminescence assay for TNF-α, IL-6, and IL-1β (Meso Scale Diagnostics, Rockville, MD, USA), according to the manufacturer’s instructions. Measurement of NF-κB transcriptional activity in the colon NF-κB activity in the colon was determined using the TransAM transcriptional factor assaying kit for NF-κB p65 (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s instructions. NF-κB p65 protein (Active Motif) was used as the standard. Statistical analyses Significance of the differences between two groups was evaluated using the F-test, followed by the Student’s or Aspin–Welch t -test. Differences in disease severity were evaluated using the Mann–Whitney U test. Declarations Ethical statement All experiments adhered to the ARRIVE guidelines and Guidelines for Animal Experimentation of the Central Pharmaceutical Research Institute of Japan Tobacco Inc. (Protocol No. 02950, Data: Jan 5, 2022). All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Central Pharmaceutical Research Institute of Japan Tobacco Inc., and all procedures were conducted in accordance with the relevant guidelines and regulations. Conflicts of interest All authors are employees of Japan Tobacco Inc. Funding Not applicable. Author Contribution SE participated in the design of the study. Most experiments were performed by SE. SE, YK and RK evaluated prednisolone, tofacitinib, and anti-TNF-α antibody in the colitis model as shown in supplementary figures. The manuscript was drafted by SE and was commented on and revised by NK. All authors have read and approved the final manuscript. Acknowledgement The authors appreciate Koji Inagaki for their histological assistance. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Gomollón, F. et al. Inflammatory bowel disease (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC), are chronic relapsing disorders of the gastrointestinal tract that are characterized pathologically by intestinal inflammation and epithelial injury. J. Crohns Colitis . 11 , 3–25 (2017). Magro, F. et al. Third European Evidence-based Consensus on Diagnosis and Management of Ulcerative Colitis. 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02:10:07","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129860,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/281d2695fbe6709eb9abbe67.html"},{"id":94612565,"identity":"4b65a38f-06f6-48e5-b1ac-67c585048096","added_by":"auto","created_at":"2025-10-29 02:11:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4350002,"visible":true,"origin":"","legend":"\u003cp\u003eInvolvement of immunoglobulin production in chronic DSS-induced colitis. (A) Experimental protocol. Mice were fed with three cycles of 1.5% DSS for seven days followed by DW \u003cem\u003ead libitum\u003c/em\u003e for seven days, after which they were administered DW for seven days. Arrows indicate the analysis time. (B) Relative weight curve of the DW (open circle)- and DSS (closed circle)-treated groups. Data are represented as mean ± SD. n = 10 animals per group. (C) Total IgG, (D) anti-flagellin IgG, (E) pANCA, and (F) total IgA levels measured by ELISA. Results are shown as mean ± SD with individual data. n = 6 animals per group. *p \u0026lt; 0.05 and **p \u0026lt; 0.01 vs. DW-treated group determined by Student’s or Aspin–Welch \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/41e823a2ff18f5dc4ac0d4c5.jpg"},{"id":94602901,"identity":"3381f58a-a0c0-40d3-ac27-1fefcde4782a","added_by":"auto","created_at":"2025-10-28 20:05:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3038480,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation of IgG-producing plasma cells with colitis onset. (A) Numbers of infiltrating inflammatory cells, including CD4 T cells (CD4T), CD8 T cells (CD8T), B cells (B), macrophages (Mφ), dendritic cells (DCs), inflammatory monocytes (Mono), and neutrophils (Neu), in DW (white column)- and DSS (black column)-treated groups at the experimental endpoint (day\u0026nbsp;50) determined by flow cytometry. (B) Immunofluorescence double staining of the colon of DW- and DSS-treated groups with anti-CD19 (green) and anti-IgG (red) at the experimental endpoint. DAPI (white). Double-positive cells displaying CD19\u003csup\u003e+\u003c/sup\u003eIgG\u003csup\u003e+\u003c/sup\u003e are observed in yellow (arrowheads). Data are representative of five mice per group. Scale bar, 100 µm. (C) Intracellular immunoglobulin staining of B220\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e immature plasma cells and B220\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B cells at the experimental endpoin. In the expression analysis of CD138, gray histogram indicates the isotype control staining. Data are representative of five DSS-treated mice. (D) Total plasma, (E) IgA-producing, and (F) IgG2-producing plasma cell numbers were determined. (A, D, E, and F) Results are shown as mean ± SD with individual data. n\u0026nbsp;=\u0026nbsp;5–6 animals per group. *p\u0026nbsp;\u0026lt;\u0026nbsp;0.05 and **p\u0026nbsp;\u0026lt;\u0026nbsp;0.01 vs. DW-treated group determined by Student’s or Aspin–Welch \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/89c7098a0fb9712b56e76803.jpg"},{"id":94602900,"identity":"fca098f5-606e-484b-8253-63a9dcc2e00b","added_by":"auto","created_at":"2025-10-28 20:05:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2492610,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of bortezomib on plasma IgG levels and IgG-producing immature plasma cells in the colon of chronic DSS-induced colitis model mice. (A) Experimental design of bortezomib administration process. Arrow indicates the duration of bortezomib administration. (B) Total IgG and (C) pANCA levels measured by ELISA. (D) Numbers of infiltrating inflammatory cells, including CD4 T cells (CD4T), CD8 T cells (CD8T), B cells (B), plasma cells, macrophages (Mφ), dendritic cells (DCs), inflammatory monocytes (Mono), and neutrophils (Neu), in sham-treated (white column), pre-treatment (dotted column), vehicle-treated (black column), and bortezomib-treated (grey column) groups determined by flow cytometry. (E) IgG2- and (F) IgA-producing immature plasma cell number were determined by flow cytometry. (G) Representative microphotographs of IgG deposits in the colonic tissues of sham-, vehicle-, and bortezomib-treated mice. IgG (red); DAPI (blue). Scale bar, 100 µm. (H) Percentage of IgG-positive area in the distal colon. (B−F and H) Results are shown as mean ± SD with individual data. *p \u0026lt; 0.05 and **p \u0026lt; 0.01 vs. sham-treated group; §p \u0026lt; 0.05 and §§p \u0026lt; 0.01 vs. pre-treatment group; #p \u0026lt; 0.05 and ##p \u0026lt; 0.01 vs. vehicle-treated group (Student’s or Aspin–Welch \u003cem\u003et\u003c/em\u003e-test). n = 5–7 animals per group.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/51fb523356d103f5fabe6e26.jpg"},{"id":94612579,"identity":"1209bb57-1e70-4349-9ef7-e33df82bb093","added_by":"auto","created_at":"2025-10-29 02:11:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1629565,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of bortezomib on colitis progression and intestinal fibrosis in the distal colon of chronic DSS-induced colitis model mice.\u003cstrong\u003e \u003c/strong\u003e(A) Representative microphotographs of the colonic tissues of sham-treated, pre-treatment, vehicle-treated, and bortezomib-treated mice (H\u0026amp;E staining). Scale bar, 100 µm. (B) Histological scores of inflammatory infiltration, crypt epithelial changes including epithelial hyperplasia, goblet cell loss, crypt loss, cryptitis, and crypt abscess and total score at the experimental end point. Results are shown as mean ± SD with individual data. **p \u0026lt; 0.01 vs. sham-treated group; #p \u0026lt; 0.05 vs. vehicle-treated group (Mann–Whitney \u003cem\u003eU\u003c/em\u003e test). n = 5–7 animals per group. (C) Representative microphotographs of the colonic tissues of sham-treated, pre-treatment, vehicle-treated, and bortezomib-treated mice (Masson’s trichrome staining). Scale bar, 100 µm. (D) Percentage of Masson-positive area in the distal colon. **p \u0026lt; 0.01 vs. sham-treated group (Aspin–Welch \u003cem\u003et\u003c/em\u003e-test). Results are shown as mean ± SD with individual data. §p \u0026lt; 0.05 vs. pre-treatment group (Student’s \u003cem\u003et\u003c/em\u003e-test). ##p \u0026lt; 0.01 vs. vehicle-treated group (Student’s \u003cem\u003et\u003c/em\u003e-test). n = 5–7 animals per group. (E) Representative microphotographs of the colonic tissues of sham-, vehicle-, and bortezomib-treated mice at the experimental endpoint. αSMA (red); DAPI (blue). Scale bar, 50 µm.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/d21b1336ba8f1c1ab0ea641c.jpg"},{"id":100070850,"identity":"d41d67a6-7429-4225-9f9a-06a5f41509dc","added_by":"auto","created_at":"2026-01-12 16:18:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12425509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/48c8d3a3-0ce7-438b-8e22-c3a8ff93c719.pdf"},{"id":94602898,"identity":"c06fb494-77f5-414e-8a32-78fca7c0ac82","added_by":"auto","created_at":"2025-10-28 20:05:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":610304,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7646295/v1/321db67b2a77b370ecb4d338.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Depletion of IgG-producing plasma cells in the colon by treatment of proteasome inhibitor ameliorates chronic DSS-induced colitis in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn\u0026rsquo;s disease, is a group of chronic relapsing gastrointestinal tract disorders pathologically characterized by intestinal inflammation and epithelial injury [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Basal plasmacytosis, defined as dense infiltration of plasma cells around the deep part of the lamina propria (LP) or at the base of colon crypts, is a histological feature of IBD, particularly UC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. High degree of basal plasmacytosis is associated with high clinical relapse rate [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], shorter clinical relapse time [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and elevated fecal calprotectin levels (disease activity markers) in patients with UC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor patients with UC, several antibodies are used as serological markers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Specifically, perinuclear anti-neutrophil cytoplasmic antibody (pANCA), an autoantibody, is the best marker for UC in terms of sensitivity and specificity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, autoantigens, such as integrin αvβ6 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and endothelial protein C receptor [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], have also been identified in patients with UC. Serum levels of IgG [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and pANCA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] positively correlate with the Mayo Clinic score, suggesting that IgG-producing plasma cells are associated with UC pathogenesis. Plasma cells comprised one of the major cellular components in the colonic LP of patients with UC [14\u0026thinsp;\u0026minus;\u0026thinsp;16]. In a healthy state, most plasma cells are IgA-producing plasma cells; however, marked IgG-producing plasma cell infiltration is observed in the colon of patients with UC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. IgG-producing plasma cells infiltrate the inflamed mucosa and critically influence UC pathogenesis by exacerbating mucosal inflammation by activation of pathogenic intestinal macrophages via Fc gamma receptor (FcγR) signaling [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Despite these insights, the precise roles of IgG-producing plasma cells in UC remain unclear [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChronic dextran sulfate sodium (DSS)-induced colitis model is established by administering three repeated cycles of \u003cem\u003ead libitum\u003c/em\u003e of DSS followed by distilled water (DW) every seven days [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The chronic DSS-induced colitis model exhibits more resemblance to chronic UC than the acute DSS-induced colitis model in terms of the association with both myeloid cells, such as macrophages and neutrophils, and lymphocytes, such as T and B cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the chronic colitis model is often used to evaluate the therapeutic efficacy of anti-inflammatory drugs for UC, such as glucocorticoids (dexamethasone) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], Janus kinase (JAK) inhibitors (tofacitinib) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and anti-tumor necrosis factor (TNF)-α antibodies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBortezomib is a proteasome inhibitor that enhances the accumulation of unfolded proteins with the induction of endoplasmic reticulum (ER) stress, leading to apoptotic cell death [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Plasma cells synthesizing high levels of immunoglobulins exhibit relatively high sensitivity to bortezomib [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], contributing to the clinical success of bortezomib for multiple myeloma treatment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Bortezomib ameliorates lupus nephritis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], ANCA-induced glomerulonephritis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and myasthenia gravis (MG) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] in rodents by inhibiting the production of autoantibodies by plasma cell depletion. Additionally, bortezomib protects against autoimmune diseases, such as systemic lupus erythematosus [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], thrombotic thrombocytopenic purpura [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], MG [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and neuromyelitis optica spectrum disorder [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], by reducing the plasma cell number and suppressing autoantibody production.\u003c/p\u003e\u003cp\u003eIn this study, we established a chronic colitis model by repeated DSS treatment of mice and investigated the roles of IgG-producing plasma cells in colitis pathogenesis. Additionally, to demonstrate plasma cells as causative cells for chronic colitis, we examined the effects of bortezomib, which depletes plasma cells, in a colitis model.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003epANCA and anti-flagellin IgG are produced during intestinal inflammation in chronic DSS-induced colitis model mice\u003c/h2\u003e\u003cp\u003eChronic colitis was induced by administering three repeated cycles of DSS. Each cycle involved the administration of 1.5% DSS for seven days, followed by sterile DW \u003cem\u003ead libitum\u003c/em\u003e for seven days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the DSS-induced colitis model, mice treated with DSS started to exhibit clinical signs of colitis, including weight loss with loose stools, diarrhea, and hematochezia, after five days of DSS administration during the first DSS/DW cycle. Body weight further decreased when DW was reintroduced on day 8, with maximal weight loss observed on day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Colitis model mice started to regain weight at the end of the first DSS/DW cycle (day 15) until the initial body weight was reached at the end of the experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, after the first DSS/DW cycle, during the course of acute intestinal inflammation, total IgG levels in the plasma were lower than those in DW-treated mice, consistent with a previous report [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, after the second DSS/DW cycle during the chronic inflammation phase, total IgG levels were higher than those in DW-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Next, we examined whether IgG against flagellin, protein subunit of bacterial flagellum, or myeloperoxidase (MPO), an autoantigen, were present in the chronic DSS-induced colitis model mice during intestinal inflammation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, although the plasma flagellin-specific IgG antibody levels increased only after the second DSS/DW cycle (day 29), consistent with previous reports [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], these antibody levels did not increase during the chronic phase. In contrast, pANCA (anti-MPO IgG) levels consecutively increased after the second DSS/DW cycle until the experimental endpoint (day 50; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Plasma IgA levels did not increase at any time point, indicating that they did not affect colitis development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Taken together, these results suggest that autoantibodies, such as pANCA, are involved in colitis pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIgG-producing plasma cells infiltrate into colonic LP in chronic DSS-induced colitis model mice\u003c/h3\u003e\n\u003cp\u003eFlow cytometric analysis revealed significantly higher numbers of CD4 T cells (TCRβ\u003csup\u003e+\u003c/sup\u003eCD335\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), B cells (CD19\u003csup\u003e+\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003e), macrophages (CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), inflammatory monocytes (CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e), and neutrophils (CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) in the colonic LP of DSS-treated mice than in that of DW-treated mice at the experimental endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;1). Furthermore, consistent with the increase in total plasma IgG and pANCA production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and E), IgG-expressing CD19\u003csup\u003e+\u003c/sup\u003e cells were observed in the mucosa and submucosa of the colon of DSS-treated mice at the experimental endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We confirmed the infiltration of CD19\u003csup\u003e+\u003c/sup\u003e cells into the colonic LP of the chronic DSS-induced colitis model using flow cytometry. CD19\u003csup\u003e+\u003c/sup\u003e cells infiltrating the colon were B220\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e B and B220\u003csup\u003e\u0026minus;\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003eCD138\u003csup\u003e+\u003c/sup\u003e immature plasma cells (also called plasmablasts; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Low CD138 levels in immature plasma cells are possibly due to its instability, which affects its detection during collagenase digestion [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, immature plasma cells expressed IgA and IgG2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, B cells mildly expressed IgA, but not IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition to the various inflammatory cells, the number of immature plasma cells also increased in the colon of the chronic DSS-induced colitis model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F, both IgA- and IgG-producing immature plasma cells were more abundant in DSS-treated mice than in the DW-treated mice. Overall, plasma cells infiltrating the colon in the chronic DSS-induced colitis model were IgG2-producing and exhibited an immature phenotype.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of bortezomib on the inflammatory responses in chronic DSS-induced colitis model mice\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of colon-infiltrating IgG-producing immature plasma cells in the chronic colitis progression, immature plasma cells were depleted by systemic administration of bortezomib. For intervention studies, bortezomib was administered twice weekly to the mice at the end of the second DSS/DW cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To explore the inhibitory effects of bortezomib, total IgG and pANCA levels in plasma, which were upregulated during colitis progression, were examined. Bortezomib significantly decreased the total IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and pANCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) levels at the experimental endpoint comparable to those at the start of treatment (pre-treatment). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the numbers of CD4 T cells, B cells, plasma cells, macrophages, inflammatory monocytes, and neutrophils increased at the start of bortezomib treatment. Moreover, the numbers of macrophages and neutrophils decreased, but those of other cells, excluding plasma cells, remained unchanged at the end of treatment in the vehicle group. Bortezomib significantly decreased the numbers of B and plasma cells, but not CD4 T cells, macrophages, inflammatory monocytes, and neutrophils, in the colonic LP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F, the numbers of IgG2- and IgA-producing immature plasma cells increased during colitis progression. Notably, bortezomib reduced the number of IgG2-producing immature plasma cells more than that of B or total plasma cells, but not IgA-producing immature plasma cells, in the colonic LP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Moreover, IgG was deposited in the mucosa and submucosa of the colon in the chronic DSS-induced colitis model at the experimental endpoint, similar to that observed in patients with UC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Bortezomib significantly decreased the colonic deposition of IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAmelioration of colitis and intestinal fibrosis by bortezomib in chronic DSS-induced colitis model mice\u003c/h3\u003e\n\u003cp\u003eTo investigate the effects of bortezomib on colitis pathogenesis and intestinal fibrosis, we performed histopathological evaluation of colitis (H\u0026amp;E staining) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and quantification of collagen deposition in the mucosa and submucosa of the colon (Masson\u0026rsquo;s trichrome staining) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. At the start of treatment with bortezomib (pre-treatment), the mice already exhibited colitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B) and mild intestinal fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Bortezomib ameliorated colitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B) and inhibited intestinal fibrosis progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). As shown in Supplementary Fig.\u0026nbsp;2A\u0026ndash;C, bortezomib significantly reduced the colon weight-to-length ratio and colonoscopic disease severity, including mucosal thickening, vasculature, and granularity, at the experimental endpoint. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, alpha-smooth muscle actin (αSMA) was localized not only in the typical tissue layers, such as muscolaris mucosa and muscolaris propria, but also in the mucosa of the chronic DSS-induced colitis model, similar to that observed in patients with UC [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Bortezomib decreased the αSMA levels in the colon at the experimental endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Along with the cell count results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F, these findings suggest that bortezomib ameliorates colitis and the intestinal fibrosis progression by selectively depleting the IgG-producing plasma cells, without significantly affecting the other immune cells, including innate immune and CD4 T cells. Although the production of inflammatory cytokines, such as TNF-α, interleukin (IL)-6, and IL-1β, and nuclear factor (NF)-κB activation increased with the development of chronic colitis, bortezomib had no effects on these processes (Supplementary Fig.\u0026nbsp;3). Furthermore, anti-inflammatory agents targeting the innate immune or T cells, such as glucocorticoids (prednisolone), JAK inhibitors (tofacitinib), and anti-TNF-α antibodies, did not inhibit the development of colitis in the chronic DSS-induced colitis model (Supplementary Fig.\u0026nbsp;4). Therefore, IgG-producing immature plasma cells play crucial roles in the development and progression of colitis in the chronic DSS-induced colitis model. In summary, bortezomib significantly ameliorated chronic colitis by reducing the number of colonic IgG-producing immature plasma cells in the colon of the chronic DSS-induced colitis model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn a double-blind, randomized, placebo-controlled trial (phase II trial), anti-CD20 antibody rituximab did not induce remission in moderately active UC [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Rituximab depleted B cells, but not plasmablasts/plasma cells, in the colonic mucosa of patients with UC in a clinical trial, considering that plasmablasts/plasma cells remaining at the site of inflammation continuously supported the ongoing inflammatory process of UC [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we found that the chronic DSS-induced colitis model exhibits resemblance to the chronic nature of human UC in terms of histopathological features, including IgG-producing plasma cell infiltration into the colon. Interestingly, flow cytometric analysis showed that IgG-producing plasma cells infiltrating the colonic LP of chronic DSS-induced colitis model were immature plasma cells (also called plasmablasts), previously reported in patients with UC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Antimicrobial antibodies are produced in the early stages, whereas autoantibodies are produced in the later stages of colitis. Therefore, colonic bacteria possibly contain proteins cross-reactive to the autoantigen epitope, resulting in a destructive inflammatory response triggered by the influx of luminal bacterial products into the gut wall via epithelial damage directed toward a self-antigen, leading to the progression from acute to chronic colitis in a colitis model, similar to that observed in patients with UC [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis study showed that bortezomib, a plasma cell-targeted proteasome inhibitor, significantly improved the colonoscopic and histopathological manifestations in the chronic DSS-induced colitis model. In addition to inducing ER stress, bortezomib inhibits NF-κB activation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Bortezomib also inhibits the production of inflammatory cytokine, such as IL-6, TNF-α, and IL-1β, by suppressing NF-κB activation, thereby ameliorating acute DSS-inducing colitis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, bortezomib inhibited colitis progression without suppressing inflammatory cytokine production and NF-κB activation in the chronic-phase DSS-induced colitis model in this study. Bortezomib reduced not only plasma IgG levels but also number of IgG-producing immature plasma cells and IgG deposition in the colon of chronic DSS-induced colitis model mice. Therefore, bortezomib ameliorated chronic colitis by reducing the number of IgG-producing immature plasma cells in the colon. Interestingly, bortezomib did not decrease the number of IgA-producing immature plasma cells in the colon, suggesting differential activation of the ER stress-induced unfolded protein response between IgA- and IgG-producing immature plasma cells. As IgA production at mucosal sites is necessary to maintain a non-inflammatory state and prevent inflammatory responses [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], targeting IgG-producing immature plasma cells rather than IgA-producing immature plasma cells is important to prevent colitis.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eUC is associated with progressive fibrosis of the mucosa and submucosa linked to the severity and chronicity of inflammation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], suggesting the importance of deep remission, including histological remission, as a therapeutic target for UC [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Intestinal fibrosis was also observed in our chronic DSS-induced colitis model, consistent with previous reports [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], was suppressed by bortezomib. Bortezomib significantly reduces lung fibrosis in bleomycin-treated mice by depleting the plasma cells, indicating the causal role of plasma cells in lung fibrosis development [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Thus, these findings suggest plasma cells as therapeutic targets for fibrosis, including lung and intestinal fibrosis.\u003c/p\u003e\u003cp\u003eIn contrast to previous reports [21\u0026thinsp;\u0026minus;\u0026thinsp;23], our chronic DSS-induced colitis model was resistant to treatment with glucocorticoids (prednisolone), JAK inhibitors (tofacitinib), and anti-TNF-α antibodies, possibly due to the variability in the microbiome among different facilities [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, our chronic DSS-induced colitis model was possibly a severe UC murine model that was not responsive to major therapeutic agents, as described above. Serum pANCA levels are associated with the lack of response to anti-TNF-α therapy in patients with UC [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, number of infiltrating plasma cells in the colon is higher in patients with UC non-responsive to anti-TNF agents [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and glucocorticoids (prednisolone) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] than in the responders. Therefore, plasma cell depletion may serve as an effective therapeutic approach for drug-resistant UC characterized by marked plasma cell infiltration into the colon.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrated the ameliorative effects of bortezomib, which depletes plasma cells, on chronic DSS-induced colitis. However, further translational research and clinical studies are necessary to assess the efficacy of bortezomib for UC treatment. Nevertheless, this study provides insights into the \u003cem\u003ein vivo\u003c/em\u003e functions of IgG-producing immature plasma cells in the colonic LP of patients with UC. Our findings suggest that IgG-producing immature plasma cell depletion in the colon ameliorates chronic colitis, highlighting a novel therapeutic approach for human UC.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and method","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eFemale C57BL/6J (B6J) mice (weighting 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) were purchased from CLEA Japan (Tokyo, Japan) and maintained under specific pathogen-free conditions at a room temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and air humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;15% under a 12/12 h light/dark cycle.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChronic DSS-induced colitis model establishment and treatment\u003c/h3\u003e\n\u003cp\u003eColitis was induced as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, mice were fed 1.5% DSS (36\u0026ndash;50 kDa; MP Biomedicals, Solon, OH, USA) dissolved in sterile DW (Otsuka Pharmaceutical, Tokyo, Japan) \u003cem\u003ead libitum\u003c/em\u003e for seven days, followed by DW for seven days. This DSS/DW cycle was repeated twice, after which the mice were administered DW for seven days to establish a chronic DSS-induced colitis model. Sham group was treated with DW. Bortezomib was purchased from Santa Cruz (Dallas, TX, USA), dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) at a concentration of 10 mg/mL, and suspended in PBS. Female B6J mice were mixed and caged into groups of seven mice at ten days from the initial day based on their body weight (bw). Then, the mice were intravenously treated with the vehicle alone or bortezomib (750 \u0026micro;g/kg bw) twice weekly for three weeks from the end of the second DSS/DW cycle. In another experiment, after the second DSS/DW cycle, the mice were gavaged daily with another vehicle (0.5% methylcellulose (MC)), 3 mg/kg bw prednisolone or 100 mg/kg bw tofacitinib, or administrated intraperitoneally 1000 \u0026micro;g of anti-TNF-α antibody (clone TN3-19.12; Bio X cell, Lebanon, NH, USA) or Armenian hamster IgG (Bio X cell) twice a week.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eColonoscopy and histology\u003c/h2\u003e\u003cp\u003eFor colonoscopy, the mice were anesthetized with isoflurane (Viatris, Tokyo, Japan), and feces were removed by injecting saline through a flexible feeding tube (Fuchigami, Kyoto, Japan). A rigid telescope (diameter, 1.9 mm; Smith and Nephew, London, UK) was rectally inserted into the mice up to 3 cm using the Olympus CLH-250 Xenon Light Source (Olympus, Tokyo, Japan), as previously described [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. During endoscope withdrawal, a video of the distal colon was recorded using the Olympus Video System OTV-SC2 (Olympus) and TEAC UR-4MD Medical Video Recorder (TEAC, Tokyo, Japan). Colonoscopic findings were scored as follows: Mucosal thickening (0\u0026thinsp;=\u0026thinsp;transparent, 1\u0026thinsp;=\u0026thinsp;moderate, 2\u0026thinsp;=\u0026thinsp;marked, and 3\u0026thinsp;=\u0026thinsp;non-transparent), vasculature (0\u0026thinsp;=\u0026thinsp;normal, 1\u0026thinsp;=\u0026thinsp;moderate, 2\u0026thinsp;=\u0026thinsp;marked, and 3\u0026thinsp;=\u0026thinsp;absent/bleeding), and granularity (0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;moderate, 2\u0026thinsp;=\u0026thinsp;marked, and 3\u0026thinsp;=\u0026thinsp;extreme) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Colonoscopy findings were scored by a blinded reviewer. For microscopic examination, colons were removed within 24 h of endoscopy after euthanizing the mice via cervical dislocation under anesthesia with isoflurane (Viatris). Distal colons (3 cm) were longitudinally cut, fixed with 10% neutral-buffered formalin (FUJIFILM Wako Pure Chemical, Osaka, Japan), embedded in paraffin wax, and stained with hematoxylin and eosin (H\u0026amp;E). Tissue specimens for pathological evaluation were prepared by the Biopathology Institute (Oita, Japan). Distal colons were scored according to the histological criteria as follows: Inflammatory infiltration (0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;10% mucosal infiltration, 2\u0026thinsp;=\u0026thinsp;10\u0026ndash;25% mucosal infiltration and little submucosal infiltration, 3\u0026thinsp;=\u0026thinsp;26\u0026ndash;50% submucosal infiltration, and 4\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;50% transmural infiltration), epithelial hyperplasia (0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;1.25-fold increase in epithelial length, 2\u0026thinsp;=\u0026thinsp;1.25\u0026ndash;1.35-fold increase in epithelial length, 3\u0026thinsp;=\u0026thinsp;1.36\u0026ndash;1.5-fold increase in epithelial length, and 4\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;1.5-fold increase in epithelial length), goblet cell and crypt loss (0\u0026thinsp;=\u0026thinsp;none, 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;20%, 2\u0026thinsp;=\u0026thinsp;20\u0026ndash;35%, 3\u0026thinsp;=\u0026thinsp;36\u0026ndash;50%, and 4\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;50%), cryptitis (0\u0026thinsp;=\u0026thinsp;none and 2\u0026thinsp;=\u0026thinsp;presence), and crypt abscess (0\u0026thinsp;=\u0026thinsp;none and 3\u0026thinsp;=\u0026thinsp;presence) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Histological scoring was supported by KAC (Kyoto, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of plasma antibody levels\u003c/h2\u003e\u003cp\u003ePlasma samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Plasma IgG and IgA levels were determined using ELISA kits (Bethyl Laboratories, Montgomery, TX, USA), according to the manufacturer\u0026rsquo;s instructions. Levels of pANCA (anti-MPO IgG) and anti-flagellin IgG antibodies were detected by ELISA. Briefly, diluted plasma samples and standards (anti-MPO IgG (Abcam, Cambridge, UK) or anti-flagellin IgG (InvivoGen, San Diego, CA, USA)) were added to wells pre-coated with recombinant mouse MPO (R\u0026amp;D Systems, Minneapolis, MN, USA) or flagellin (FLA-ST; InvivoGen) and incubated at room temperature for 2 h. After washing with PBS/Tween 20, diluted horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and incubated for 2 h. After washing with PBS/Tween 20, TMB substrate was added to each well and incubated in the dark. Subsequently, the stop solution was added to each well, and absorbance was measured at 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIsolation of colonic LP cells\u003c/h2\u003e\u003cp\u003eLP cells were isolated from the colon, as previously described [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Briefly, distal colons were longitudinally cut, washed with cold Hank\u0026rsquo;s balanced salt solution containing 2% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 U/mL), and minced. The tissues were completely digested at 37\u0026deg;C for 40 min with gentle stirring in the RPMI-1640 medium containing 2% FBS, penicillin (100 U/mL), and streptomycin (100 U/mL) and supplemented with collagenase D (100 U/mL; Sigma-Aldrich, St Louis, MO, USA) and DNase (20 mg/mL; Roche Diagnostics, Basel, Switzerland). LP cells were purified on a 40/75% Percoll gradient via centrifugation at 600 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 25\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eAntibodies against CD45, TCRβ, CD11b, B220, CD19, CD138, and IgA were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against Ly6G, CD4, CD8b, CD11b, CD11c, F4/80, Ly6G, Ly6C, CD335 (NKp46), IgG1, IgG2a/b, and IgG3 were purchased from BioLegend (San Diego, CA, USA). Cell surface staining was performed according to standard techniques after treatment with the anti-CD16/32 antibody (BD Biosciences) to block FcγR binding. Dead cells were excluded using the Fixable Viability Dye eFluor 780 (FVD780; Thermo Fisher Scientific). For intracellular staining, the cells were stained with different cell surface antibodies, fixed, permeabilized, and intracellularly stained for IgA, IgG1, IgG2a/b, or IgG3. Gating strategies were set with reference to the isotype or fluorescence minus one control. Flow cytometry was performed using the BD LSRFortessa X-20 flow cytometer (BD Biosciences), and data were analyzed using the FlowJo version 10.8.0 software (BD Biosciences).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry for IgG\u003c/h2\u003e\u003cp\u003eFor CD19 and IgG staining, deparaffinized and rehydrated sections were treated with a protease solution (Nichirei Biosciences, Tokyo, Japan) for 15 min at 37\u0026deg;C and incubated with Blocking One (Nacalai Tesque) for 1 h at room temperature. Then, the sections were incubated with Alexa Fluor 488-conjugated anti-CD19 (BioLegend) and Alexa fluor 555-conjugated anti-IgG (BioLegend) at 4\u0026deg;C overnight. Nuclei were counterstanied using 4',6-diamidino-2-phenylindole (DAPI; SouthernBiotech, Birmingham, AL, USA). The sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the FV3000 confocal laser-scanning microscope (Olympus) for double-positive cells. For IgG deposits, the sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the BZ-X800 fluorescence microscope (Keyence, Osaka, Japan). Microscopic images were digitized, and IgG-positive areas were determined by red\u0026ndash;green\u0026ndash;blue (RGB) segmentation. Digital images were analyzed using inform version 3.1.2 software (PerkinElmer, Hopkinton, MA, USA). Results were calculated as the number of IgG-positive areas in the mucosa and submucosa in all fields of the distal colon.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eColonic fibrosis analysis\u003c/h2\u003e\u003cp\u003eMasson\u0026rsquo;s trichrome staining was performed to measure the collagen deposition as fibrosis. The tissue specimens were prepared for pathological evaluation at the Biopathology Institute. Microscopic images were digitized, and collagen-positive areas were determined by RGB segmentation. Digital images were analyzed using the inform version 3.1.2 software (PerkinElmer). Results were calculated as the number of collagen-positive areas in the mucosa and submucosa in all fields of the distal colon. For αSMA staining, deparaffinized and rehydrated sections were treated with HISTOFINE (pH 9.0; Nichirei Biosciences) for 15 min at 95\u0026deg;C and incubated with Blocking One (Nacalai Tesque) for 30 min at room temperature. Then, αSMA staining was performed using rabbit anti-αSMA (Abcam) at 4\u0026deg;C overnight, followed by incubation with Alexa Fluor 647 anti-rabbit IgG (Abcam) at room temperature for 1 h. Nuclei were counterstained with DAPI (SouthernBiotech). The sections were mounted using Fluoromount-G (SouthernBiotech) and sequentially scanned using the BZ-X800 fluorescence microscope (Keyence).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of colonic cytokine levels\u003c/h2\u003e\u003cp\u003eDistal colonic segments were rinsed with saline, blotted dry, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. The segments were homogenized using beads in DW containing the Sample Diluent Concentrate 1 (R\u0026amp;D Systems) and protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 12000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C to remove the debris, protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA). Colonic cytokine levels were quantified using the Meso Scale Discovery electrochemiluminescence assay for TNF-α, IL-6, and IL-1β (Meso Scale Diagnostics, Rockville, MD, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of NF-κB transcriptional activity in the colon\u003c/h2\u003e\u003cp\u003eNF-κB activity in the colon was determined using the TransAM transcriptional factor assaying kit for NF-κB p65 (Active Motif, Carlsbad, CA, USA), according to the manufacturer\u0026rsquo;s instructions. NF-κB p65 protein (Active Motif) was used as the standard.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eSignificance of the differences between two groups was evaluated using the F-test, followed by the Student\u0026rsquo;s or Aspin\u0026ndash;Welch \u003cem\u003et\u003c/em\u003e-test. Differences in disease severity were evaluated using the Mann\u0026ndash;Whitney U test.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical statement\u003c/h2\u003e\u003cp\u003e All experiments adhered to the ARRIVE guidelines and Guidelines for Animal Experimentation of the Central Pharmaceutical Research Institute of Japan Tobacco Inc. (Protocol No. 02950, Data: Jan 5, 2022). All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Central Pharmaceutical Research Institute of Japan Tobacco Inc., and all procedures were conducted in accordance with the relevant guidelines and regulations.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eAll authors are employees of Japan Tobacco Inc.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSE participated in the design of the study. Most experiments were performed by SE. SE, YK and RK evaluated prednisolone, tofacitinib, and anti-TNF-α antibody in the colitis model as shown in supplementary figures. The manuscript was drafted by SE and was commented on and revised by NK. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciate Koji Inagaki for their histological assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGomoll\u0026oacute;n, F. et al. Inflammatory bowel disease (IBD), such as Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), are chronic relapsing disorders of the gastrointestinal tract that are characterized pathologically by intestinal inflammation and epithelial injury. \u003cem\u003eJ. Crohns Colitis\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, 3\u0026ndash;25 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMagro, F. et al. 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Macrophage polarization toward M1 phenotype in T cell transfer colitis model. \u003cem\u003eBMC Gastroenterol.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 411 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"IgG-producing plasma cells, chronic DSS-induced colitis model, proteasome inhibitor, pANCA, ulcerative colitis","lastPublishedDoi":"10.21203/rs.3.rs-7646295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7646295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBasal plasmacytosis is a histopathological hallmark of ulcerative colitis (UC). However, the precise roles of plasma cells in UC pathogenesis remains unknown. In this study, we investigated the effects of a proteasome inhibitor, which depletes plasma cells, on chronic colitis progression in mice to clarify their contribution to disease pathogenesis. Chronic colitis was induced in female C57BL/6 mice by three cycles of ad libitum dextran sulfate sodium (DSS) feeding followed by distilled water (DW), each cycle lasting seven days. The proteasome inhibitor bortezomib was administered intravenously twice weekly for three weeks after the second DSS/DW cycle. Elevated plasma IgG and perinuclear anti-neutrophil cytoplasmic antibody (pANCA) levels, as well as infiltration of IgG-producing plasma cells into the colon, were observed after the second DSS/DW cycle in chronic DSS-induced colitis model mice. Plasma cells in the colon exhibited an immature CD19⁺CD138⁺ phenotype. Bortezomib significantly ameliorated colitis and intestinal fibrosis by reducing plasma IgG and pANCA levels and the number of IgG-producing plasma cells in the colon. In conclusion, IgG-producing plasma cells were involved in colitis pathogenesis, and their depletion ameliorated colitis. Thus, IgG-producing plasma cells are causative factors and potential therapeutic targets for UC.\u003c/p\u003e","manuscriptTitle":"Depletion of IgG-producing plasma cells in the colon by treatment of proteasome inhibitor ameliorates chronic DSS-induced colitis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 20:05:19","doi":"10.21203/rs.3.rs-7646295/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T10:17:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T01:24:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T06:32:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T20:06:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143101922737041653650334616174136267384","date":"2025-10-19T06:14:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T14:17:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239912331173001913164532165823186200915","date":"2025-10-16T10:39:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186797881115477852378153182811264531733","date":"2025-10-16T06:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152931506358912130051447460946517989044","date":"2025-10-14T19:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214000217057165278967277775749968316515","date":"2025-10-14T18:54:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278396279052387028711853120321287048473","date":"2025-10-14T12:48:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T04:54:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T02:30:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-29T09:06:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-29T08:49:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0d6c79f-540c-42d3-865d-eb3127b3908d","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56811988,"name":"Health sciences/Diseases"},{"id":56811989,"name":"Health sciences/Gastroenterology"},{"id":56811990,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-01-12T16:15:22+00:00","versionOfRecord":{"articleIdentity":"rs-7646295","link":"https://doi.org/10.1038/s41598-025-34868-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-08 15:58:01","publishedOnDateReadable":"January 8th, 2026"},"versionCreatedAt":"2025-10-28 20:05:19","video":"","vorDoi":"10.1038/s41598-025-34868-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34868-6","workflowStages":[]},"version":"v1","identity":"rs-7646295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7646295","identity":"rs-7646295","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}